Antifungal susceptibility testing method for resource constrained laboratories.

PURPOSE: In resource-constrained laboratories of developing countries determination of antifungal susceptibility testing by NCCLS/CLSI method is not always feasible. We describe herein a simple yet comparable method for antifungal susceptibility testing. METHODS: Reference MICs of 72 fungal isolates including two quality control strains were determined by NCCLS/CLSI methods against fluconazole, itraconazole, voriconazole, amphotericin B and cancidas. Dermatophytes were also tested against terbinafine. Subsequently, on selection of optimum conditions, MIC was determined for all the fungal isolates by semisolid antifungal agar susceptibility method in Brain heart infusion broth supplemented with 0.5% agar (BHIA) without oil overlay and results were compared with those obtained by reference NCCLS/CLSI methods. RESULTS: Comparable results were obtained by NCCLS/CLSI and semisolid agar susceptibility (SAAS) methods against quality control strains. MICs for 72 isolates did not differ by more than one dilution for all drugs by SAAS. CONCLUSIONS: SAAS using BHIA without oil overlay provides a simple and reproducible method for obtaining MICs against yeast, filamentous fungi and dermatophytes in resource-constrained laboratories.

Detection of biofilm formation among the clinical isolates of Staphylococci: an evaluation of three different screening methods.

PURPOSE: The purpose of this study was to evaluate three methods for detection of biofilm formation in staphylococci. METHODS: For detection of biofilm formation, 152 clinical isolates of Staphylococcus spp. were screened by tissue culture plate (TCP), Tube method (TM) and Congo red agar (CRA) method. RESULTS: Of the 152 Staphylococcus spp. 88(57.8%) displayed a biofilm-positive phenotype under the optimized conditions in the TCP method and strains were further classified as high 22 (14.47 %) and moderate 60 (39.4 %) while in 70 (46.0 %) isolates weak or no biofilm was detected. Though TM correlated well with the TCP test for 18 (11.8 %) strongly biofilm producing strains, weak producers were difficult to discriminate from biofilm negative isolates. Screening on CRA does not correlate well with either of the two methods for detecting biofilm formation in staphylococci. CONCLUSION: The TCP method was found to be most sensitive, accurate and reproducible screening method for detection of biofilm formation by staphylococci and has the advantage of being a quantitative model to study the adherence of staphylococci on biomedical devices.

Evaluation of methods for AmpC beta-lactamase in gram negative clinical isolates from tertiary care hospitals.

The purpose of this study was to simultaneously screen for Extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases in gram negative clinical isolates from four tertiary care hospitals and further to compare two detection methods three-dimensional extraction method and AmpC disk test for AmpC beta-lactamases. A total of 272 isolates were screened for ESBL and AmpC beta-lactamase by modified double disk approximation method (MDDM). Synergy observed between disks of ceftazidime/cefotaxime and clavulanate were considered as ESBL producer. Isolates showing reduced susceptibility to either of the test drugs (ceftazidime or cefotaxime) and cefoxitin were considered as presumptive AmpC producers and further confirmed by three-dimensional extraction method and AmpC disk test. A total of 173 (64%) of the isolates were found to be ESBL positive and 61 (23%) showed resistant to cefoxitin. ESBL was detected in 80 (62%) isolates of E. coli and 71 (73%) of Klebsiella spp. The occurrence of AmpC beta-lactamases was found to be 8% (22) of the total isolates and the two detection methods for AmpC beta-lactamase showed concordant results. Screening for ESBL and AmpC can be simultaneously done by MDDM method and confirmation for AmpC beta-lactamase should be carried out routinely in tertiary care hospitals by AmpC disk test, as it is a simple and rapid procedure.